Project summary Mixed lineage leukemia protein-1 (MLL1) is a member of the human SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which include MLL1-4 and SETd1A/B. Rearrangements of MLL1 are frequently present in acute leukemia, whereas genetic alterations in other SET1 family members are associated with developmental disorders as well as a number of cancers. A minimal evolutionarily conserved complex, which is formed by MLL1 and four additional proteins, is required for the sequential mono- and dimethylation of H3K4. WD40 repeat protein-5 (WDR5), one of the MLL1 core complex proteins, specifically interacts with a conserved WDR5 interaction motif of the SET1 proteins, also named the Win motif. Targeting the Win motif-WDR5 interaction with small-molecule drugs and Win-based peptidomimetics has emerged as a strategic approach for treatment of acute leukemia that harbors the MLL1 protein, because the MLL1-WDR5 interaction is a key regulatory mechanism of the MLL1 enzymatic activity. However, progress in identifying inhibitors of the MLL1-WDR5 interactions remains modest due to: (i) the lack of proteomics technologies for the quantitative evaluation of the transient protein-protein interactions (PPI) at the MLL1-WDR5 interface; (ii) the lack of a mechanistic knowledge pertaining to the MLL1-WDR5 recognition system. To address these scientific and technological gaps, we will develop monomeric protein-pore based sensors for sampling transient PPI at single-recognition event resolution. The central player of these sensors will be the t-FhuA protein pore, a heavily truncated derivative of ferric hydroxamate uptake component A (FhuA) of E. coli. t-FhuA will be fused to a water- soluble MLL1 SET binding domain via a short peptide tether. Such a MLL1 binding polypeptide-containing t-FhuA-based sensor will rely on precise protein engineering, along with biomolecular recognition, scalable high-resolution electrical recordings, and single-protein channel reconstitution. The presence of WDR5 will produce a specific, sensitive, and quantitative readout that encompasses reversible current blockades, the nature of which depends on the PPI strength and WDR5 concentration. The expected immediate outcomes will be the following: (i) the design, creation, and optimization of the next-generation t-FhuA-based sensors equipped with single receptor elements for the real-time, selective sampling of transient PPI in aqueous phase; (ii) the development of a mechanistic and quantitative information on the Win motif-WDR5 interactions for each SET1 family member; (iii) the multiplexed screening of Win motif- based inhibitors with improved translational potential. These research studies will ultimately lead to a fundamental basis for accelerated discoveries in clinical molecular diagnostics, proteomics, and biosensor technology.